Conjugated linoleic acid compositions

ABSTRACT

Novel compositions containing conjugated linoleic acids are efficacious as animal feed additives and human dietary supplements. Linoleic acid is converted to its conjugated forms in which the resulting composition is low in certain unusual isomers compared to conventional conjugated linoleic products. In addition, the inventions provides compositions that are prepared according to a novel method that controls oxidation of CLA into volatile organic compounds as well as containing metal oxidant chelators to control oxidation during storage.

RELATED APPLICATIONS

The application is a continuation in part of U.S. Ser. No. 09/132,593,filed Aug. 11, 1998 and U.S. Ser. No. 09/270,940, filed Mar. 17, 1999,which is a continuation-in-part of U.S. Ser. No. 09/042,767, filed Mar.17, 1998, now U.S. Pat. No. 6,015,833, and U.S. Ser. No. 09/042,538,filed Mar. 17, 1998 now abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of human and animalnutrition, and in particular to certain novel compositions of conjugatedlinoleic acids (CLA). These compositions are prepared according to anovel method that controls oxidation of CLA into volatile organiccompounds and in some cases contain antioxidants that control oxidation.

BACKGROUND OF THE INVENTION

In 1978, researchers at the University of Wisconsin discovered theidentity of a substance contained in cooked beef that appeared toinhibit mutagenesis. The substance was found to be a mixture ofpositional isomers of linoleic acid (C18:2) having conjugated doublebonds. The c9,t11 and t10,c12 isomers are present in greatest abundance,but it is uncertain which isomers are responsible for the biologicalactivity observed. It has been noted from labelled uptake studies thatthe 9,11 isomer appears to be somewhat preferentially taken up andincorporated into the phospholipid fraction of animal tissues, and to alesser extent the 10,12 isomer. (Ha, et al., Cancer Res., 50: 1097[1990]).

The biological activity associated with conjugated linoleic acids(termed CLA) is diverse and complex. At present, very little is knownabout the mechanisms of action, although several preclinical andclinical studies in progress are likely to shed new light on thephysiological and biochemical modes of action. The anticarcinogenicproperties of CLA have been well documented. Administration of CLAinhibits rat mammary tumorigenesis, as demonstrated by Birt, et al,Cancer Res., 52: 2035s [1992]. Ha, et al., Cancer Res., 50: 1097 [1990]reported similar results in a mouse forestomach neoplasia model. CLA hasalso been identified as a strong cytotoxic agent against target humanmelanoma, colorectal and breast cancer cells in vitro. A recent majorreview article confirms the conclusions drawn from individual studies(Ip, Am. J. Clin. Nutr., 66 (6 Supp): 1523s [1997]).

Although the mechanisms of CLA action are still obscure, there isevidence that some component(s) of the immune system may be involved, atleast in vivo. U.S. Pat. No. 5,585,400 (Cook, et al., incorporatedherein by reference), discloses a method for attenuating allergicreactions in animals mediated by type I or TgE hypersensitivity byadministering a diet containing CLA. CLA in concentrations of about 0.1to 1.0 percent was also shown to be an effective adjuvant in preservingwhite blood cells. U.S. Pat. No. 5,674,901 (Cook, et al.), incorporatedherein by reference, disclosed that oral or parenteral administration ofCLA in either free acid or salt form resulted in elevation in CD-4 andCD-8 lymphocyte subpopulations associated with cell-mediated immunity.Adverse effects arising from pretreatment with exogenous tumor necrosisfactor could be alleviated indirectly by elevation or maintenance oflevels of CD-4 and CD-8 cells in animals to which CLA was administered.Finally, U.S. Pat. No. 5,430,066, incorporated herein by reference,describes the effect of CLA in preventing weight loss and anorexia byimmune stimulation.

Apart from potential therapeutic and pharmacologic applications of CLAas set forth above, there has been much excitement regarding the use ofCLA nutritively as a dietary supplement. CLA has been found to exert aprofound generalized effect on body composition, in particularredirecting the partitioning of fat and lean tissue mass. U.S. Pat. No.5,554,646 (Cook, et al.), incorporated herein by reference, discloses amethod utilizing CLA as a dietary supplement in which pigs, mice, andhumans were fed diets containing 0.5 percent CLA. In each species, asignificant drop in fat content was observed with a concomitant increasein protein mass. It is interesting that in these animals, increasing thefatty acid content of the diet by addition of CLA resulted in noincrease in body weight, but was associated with a redistribution of fatand lean within the body. Another dietary phenomenon of interest is theeffect of CLA supplementation on feed conversion. U.S. Pat. No.5,428,072 (Cook, et al., incorporated herein by reference), provideddata showing that incorporation of CLA into animal feed (birds andmammals) increased the efficiency of feed conversion leading to greaterweight gain in the CLA supplemented animals. The potential beneficialeffects of CLA supplementation for food animal growers is apparent.

Another important source of interest in CLA, and one which underscoresits early commercial potential, is that it is naturally occurring infoods and feeds consumed by humans and animals alike. In particular, CLAis abundant in products from ruminants. For example, several studieshave been conducted in which CLA has been surveyed in various dairyproducts. Aneja, et al., J. Dairy Sci., 43: 231 [1990] observed thatprocessing of milk into yogurt resulted in a concentration of CLA.(Shanta, et al., Food Chem., 47: 257 [1993]) showed that a combinedincrease in processing temperature and addition of whey increased CLAconcentration during preparation of processed cheese. In a separatestudy, Shanta, et al., J. Food Sci., 60: 695 [1995] reported that whileprocessing and storage conditions did not appreciably reduce CLAconcentrations, they did not observe any increases. In fact, severalstudies have indicated that seasonal or interanimal variation canaccount for as much as three fold differences in CLA content of cowsmilk. (See e.g., Parodi, et al, J. Dairy Sci., 60: 1550 [1977]). Also,dietary factors have been implicated in CLA content variation, as notedby Chin, et al., J. Food Camp. Anal., 5: 185 [1992]. Because of thisvariation in CLA content in natural sources, ingestion of prescribedamounts of various foods will not guarantee that the individual oranimal will receive the optimum doses to ensure achieving the desirednutritive effect.

Linoleic acid is an important component of biolipids, and comprises asignificant proportion of triglycerides and phospholipids. Linoleic acidis known as an “essential” fatty acid, meaning that the animal mustobtain it from exogenous dietary sources since it cannot beautosynthesized. Incorporation of the CLA form of linoleic acid mayresult in a direct substitution of CLA into lipid positions whereunconjugated linoleic would have migrated. However, this has not beenproven, and some of the highly beneficial but unexplained effectsobserved may even result from a repositioning of CLA within the lipidarchitecture at sites where unconjugated linoleic acid would not haveotherwise migrated. It is now clear that one source of animal CLA,especially in dairy products, comes from the biochemical action ofcertain rumen bacteria on native linoleic acid, first isomerizing thelinoleic acid to CLA, and then secreting it into the rumen cavity.Kepler, et al., J. Nutrition, 56: 1191 [1966] isolated a rumenbacterium, Butyrivibrio fibrisolvens, which catalyzes formation of9,11-CLA as an intermediate in the biohydrogenation of linoleic acid.Chin, et al., J. Nutrition, 124: 694 [1994] further found that CLA foundin the tissues of rodent was associated with bacteria, sincecorresponding germ-free rats produced no CLA.

In the development of a defined commercial source of CLA for boththerapeutic and nutritional application, a process for generating largeamounts of defined material is needed. The problem with most CLAproducts made by conventional approaches is their heterogeneity, andsubstantial variation in isoform from batch to batch. Considerableattention has been given to the fact that the ingestion of large amountsof hydrogenated oils and shortenings, instead of animal tallow, hasresulted in a diet high in trans-fatty acid content. For example,Holman, et al., PNAS, 88:4830 [1991] showed that rats fed hydrogenatedoils gave rise to an accumulation in rat liver of unusualpolyunsaturated fatty acid isomers, which appeared to interfere with thenormal metabolism of naturally occurring polyunsaturated fatty acids.These concerns were summarized in an early Editorial in Am. J. PublicHealth, 84: 722 (1974). Therefore, there exists a strong need for abiologically active CLA product of defined composition.

DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram of the alkali isomerization process of thepresent invention.

FIG. 2 is a graph of OSI values for CLA compositions in the presence ofantioxidants.

SUMMARY OF THE INVENTION

The present invention relates to the field of human and animalnutrition, and in particular to certain novel compositions of conjugatedlinoleic acids (CLA). These compositions are prepared according to anovel method that controls oxidation of CLA into volatile organiccompounds and in some embodiments contain antioxidants that controloxidation.

The present invention provides a composition comprising an isomerized(i.e., conjugated) linoleic acid moiety of high purity. The CLA moietyis not limited to any one specific CLA moiety. Several differentmoieties are contemplated by the present invention.

In some embodiments, the CLA moiety is a free fatty acid. In otherembodiments, the CLA moiety is an alkyl ester. In still furtherembodiments, the CLA moiety is a triacylglyceride.

In some embodiments of the present invention, the composition furthercomprise a metal oxidant chelator. The present invention is not limitedto any one metal oxidant chelator. A variety of metal oxidant chelatorsare contemplated by the present invention. In some embodiments, themetal oxidant chelator comprises citric acid esters. In otherembodiments, the metal oxidant chelator comprises lecithin.

The purity of the isomerized linoleic acid composition is not limited toany specific level. Several levels of purity are contemplated by thepresent invention. In some embodiments, the composition contains lessthan 100 parts per million total of volatile organic compounds. In otherembodiments, the composition contains less than 50 parts per milliontotal of volatile organic compounds. In still other embodiments, thecomposition contains less than 10 parts per million total of volatileorganic compounds. In still further embodiments, the compositioncontains less than 5 parts per million total of volatile organiccompounds.

In some embodiments, the present invention provides a food productcomprising a isomerized conjugated linoleic acid moiety of high purityand an metal oxidant chelator. The purity of the food product containingan isomerized linoleic acid composition is not limited to any specificlevel. Several levels of purity are contemplated by the presentinvention. In some embodiments, the composition contains less than 100parts per million total of volatile organic compounds. In otherembodiments, the composition contains less than 50 parts per milliontotal of volatile organic compounds. In still other embodiments, thecomposition contains less than 10 parts per million total of volatileorganic compounds. In further embodiments, the composition contains lessthan 5 parts per million total of volatile organic compounds.

The CLA moiety contained in the food product of present invention is notlimited to any one specific CLA moiety. Several different CLA moietiesare contemplated by the present invention. In some embodiments, the CLAmoiety is a free fatty acid. In other embodiments, the CLA moiety is analkyl ester. In still further embodiments, the CLA moiety is atriacylglyceride.

The present invention is not limited to any one metal oxidant chelator.A variety of metal oxidant chelators are contemplated by the presentinvention. In some embodiments, the metal oxidant chelator comprisescitric acid esters. In other embodiments, the metal oxidant chelatorcomprises lecithin.

In some embodiments, the present invention provides a food supplementcomprising a isomerized conjugated linoleic acid moiety of high purityand an metal oxidant chelator. The purity of the food product containingan isomerized linoleic acid composition is not limited to any specificlevel. Several levels of purity are contemplated by the presentinvention. In some embodiments, the composition contains less than 100parts per million total of volatile organic compounds. In otherembodiments, the composition contains less than 50 parts per milliontotal of volatile organic compounds. In still other embodiments, thecomposition contains less than 10 parts per million total of volatileorganic compounds. In further embodiments, the composition contains lessthan 5 parts per million total of volatile organic compounds.

The CLA moiety contained in the food supplement of present invention isnot limited to any one specific CLA moiety. Several different CLAmoieties are contemplated by the present invention. In some embodiments,the CLA moiety is a free fatty acid. In other embodiments, the moiety isan alkyl ester. In further embodiments, the CLA moiety is atriacylglyceride.

The present invention is not limited to any one metal oxidant chelator.A variety of metal oxidant chelators are contemplated by the presentinvention. In some embodiments, the metal oxidant chelator comprisescitric acid esters. In other embodiments, the metal oxidant chelatorcomprises lecithin.

In some embodiments, the present invention provides a method comprisingproviding a linoleic acid containing seed oil; isomerizing the linoleicacid to form conjugated linoleic acids; and treating the conjugatedlinoleic acids to obtain a CLA composition of high purity.

Several levels of purity are contemplated by the method of the presentinvention. In some embodiments, the composition less than 100 parts permillion total of volatile organic compounds. In some embodiments, theCLA composition contains less than 50 parts per million total ofvolatile organic compounds. In other embodiments, the CLA compositioncontains less than 10 parts per million total of volatile organiccompounds. In further embodiments, the CLA composition contains lessthan 5 parts per million total of volatile organic compounds.

In other embodiments of the present invention, a composition is providedcomprising a CLA moiety having a sufficiently low volatile organiccompound concentration so that the taste and smell of said compositionis not affected. In still further embodiments of the present invention,a food product is provided that comprises a conjugated linoleic acidmoiety having a sufficiently low volatile organic compound concentrationso that the taste and smell of the food product is not affected.

Definitions

As used herein, “conjugated linoleic acid” or “CLA” refers to anyconjugated linoleic acid or octadecadienoic free fatty acid. It isintended that this term encompass and indicate all positional andgeometric isomers of linoleic acid with two conjugated carbon-carbondouble bonds any place in the molecule. CLA differs from ordinarylinoleic acid in that ordinary linoleic acid has double bonds at carbonatoms 9 and 12. Examples of CLA include cis- and trans isomers (“E/Zisomers”) of the following positional isomers: 2,4-octadecadienoic acid,4,6-octadecadienoic acid, 6,8-octadecadienoic acid, 7,9-octadecadienoicacid, 8,10-octadecadienoic acid, 9,11-octadecadienoic acid and 10,12octadecadienoic acid, 11,13 octadecadienoic acid. As used herein, “CLA”encompasses a single isomer, a selected mixture of two or more isomers,and a non-selected mixture of isomers obtained from natural sources, aswell as synthetic and semisynthetic CLA.

As used herein, the term “isomerized conjugated linoleic acid” refers toCLA synthesized by chemical methods (e.g., aqeuous alkali isomerization,non-aqueous alkali isomerization, or alkali alcoholate isomerization).

As used herein, the term “conjugated linoleic acid moiety” refers to anycompound or plurality of compounds containing conjugated linoleic acidsor derivatives. Examples include, but are not limited to fatty acids,alkyl esters, and triglycerides of conjugated linoleic acid.

As used herein, it is intended that “triglycerides” of CLA contain CLAat any or all of three positions (e.g., SN-1, SN-2, or SN-3 positions)on the triglyceride backbone. Accordingly, a triglyceride containing CLAmay contain any of the positional and geometric isomers of CLA.

As used herein, it is intended that “esters” of CLA include any and allpositional and geometric isomers of CLA bound through an ester linkageto an alcohol or any other chemical group, including, but not limited tophysiologically acceptable, naturally occurring alcohols (e.g.,methanol, ethanol, propanol). Therefore, an ester of CLA or esterifiedCLA may contain any of the positional and geometric isomers of CLA.

It is intended that “non-naturally occurring isomers” of CLA include,but are not limited to c11,t13; t11,c13; t11,t13; c11,c13; c8,t10;t8,c10; t8,t10; c8,c10; and trans-trans isomers of octadecadienoic acid,and does not include t10,c12 and c9,t11 isomers of octadecadienoic acid.“Non-naturally occurring isomers” may also be referred to as “minorisomers” of CLA as these isomers are generally produced in low amountswhen CLA is synthesized by alkali isomerization.

As used herein, “low impurity” CLA refers to CLA compositions, includingfree fatty acids, alkylesters, and triglycerides, which contain lessthan 1% total 8,10 octadecadienoic acids, 11,13 octadecadienoic acids,and trans-trans octadecadienoic acids.

As used herein, “c” encompasses a chemical bond in the cis orientation,and “t” refers to a chemical bond in the trans orientation. If apositional isomer of CLA is designated without a “c” or a “t”, then thatdesignation includes all four possible isomers. For example, 10,12octadecadienoic acid encompasses c10,t12; t10,c12; t10,t12; and c10,c12octadecadienoic acid, while t10,c12 octadecadienoic acid or CLA refersto just the single isomer.

As used herein, the term “oil” refers to a free flowing liquidcontaining long chain fatty acids (e.g., CLA), triglycerides, or otherlong chain hydrocarbon groups. The long chain fatty acids, include, butare not limited to the various isomers of CLA.

As used herein, the term “physiologically acceptable carrier” refers toany carrier or excipient commonly used with oily pharmaceuticals. Suchcarriers or excipients include, but are not limited to, oils, starch,sucrose and lactose.

As used herein, the term “oral delivery vehicle” refers to any means ofdelivering a pharmaceutical orally, including, but not limited to,capsules, pills, tablets and syrups.

As used herein, the term “food product” refers to any food or feedsuitable for consumption by humans, non-ruminant animals, or ruminantanimals. The “food product” may be a prepared and packaged food (e.g.,mayonnaise, salad dressing, bread, or cheese food) or an animal feed(e.g., extruded and pelleted animal feed or coarse mixed feed).“Prepared food product” means any pre-packaged food approved for humanconsumption.

As used herein, the term “foodstuff” refers to any substance fit forhuman or animal consumption.

As used herein, the term “volatile organic compound” refers to anycarbon-containing compound which exists partially or completely in agaseous state at a given temperature. Volatile organic compounds may beformed from the oxidation of an organic compound (e.g., CLA). Volatileorganic compounds include, but are not limited to pentane, hexane,heptane, 2-butenal, ethanol, 3-methyl butanal, 4-methyl pentanone,hexanal, heptanal, 2-pentyl furan, octanal.

As used herein, the term “metal oxidant chelator” refers to anyantioxidant that chelates metals. Examples include, but are not limitedto lecithin and citric acid esters.

As used herein, the term “alcoholate catalyst” refers to alkali metalcompounds of any monohydric alcohol, including, but not limited to,potassium methylate and potassium ethylate.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention result from a highlycontrolled isomerization process, and from using the preferred startingmaterials of sunflower, safflower, or corn oil. This composition has notheretofore been obtained, for application to an industrial scale,because the conventional processes historically produce conjugatedlinoleic acids for entirely different purposes, namely, as drying oilsin the paint industry. Also, there has not been an appreciation of theimplications of the isomer content of the final product, because theanalytical methods for characterizing the fatty acids has not beenwidely available. Furthermore, the present invention provides a methodfor preventing oxidation of CLA during storage to form volatile organiccompounds.

I. Methods for Conjugating Linoleic Acids

In the older isomerization processes, some of which are still in use inmore modem format, production of the conjugated fatty acids was carriedout in aqueous alkali (generally NaOH) at high temperatures in excess of200° C. and usually at superatmospheric pressures. For example, U.S.Pat. No. 2,350,583 (Bradley) discloses an aqueous alkali processutilizing treated soaps in which both conjugation and polymerizationoccurred under rather harsh conditions at 200 to 250° C. for a period ofseveral hours. The fractions of drying oil, starting with linseed oil,were obtained by distillation (see also Br. Pat. No. 558,881 for a verysimilar process). In a variation of the process, U.S. Pat. No. 4,381,264teaches a process where a low water content reaction zone (0.5% water)contains stoichiometric base in the presence of SO₂ to obtainconjugation of the double bonds of various polyunsaturated fatty acids.The aqueous alkali process was adapted in U.S. Pat. No. 4,164,505 to acontinuous flow process in which an alkali metal hydroxide and water arecontinuously charged in a flow zone maintained at between 200 and 370°C. At these temperatures, the time of reaction should be greatlyforeshortened, but there is relatively little control over theisomerization. At the higher end of the temperature range, one skilledin the art would predict almost complete conversion to double transspecies.

Methods of producing CLA using various nonaqueous solvents and catalystshave been described in the literature. Burr (U.S. Pat. No. 2,242,230)discloses the use of solvents such as methanol, butanol, ethanol andglycol in combination with various catalysts. These reaction parametersare summarized in Table 1. With the exception of glycol, the reactionswere conducted either under reflux conditions or in sealed tubes. Thesereaction conditions result in imprecise control of two of the importantreactions parameters-temperature and pressure. Imprecise control ofthese reactions parameters is likely to lead to less than completeconjugation and the formation of undesirable isomers.

TABLE 1 Patent 2,242,230 Solvent Catalyst Temperature Time Ethanol KOH,NaOH reflux or varied higher* Butanol KOH, NaOH reflux or varied higher*Glycol KOH 195° C. varied Isoamyl KOH reflux or varied Alcohol higher*Butanol Tributyl- 140-175° C. 22 hours amine Butanol Potassium 175° C.36 hours Acetate Butanol Trisodium 175° C. 36 hours Phosphate ButanolPotassium 175° C. 36 hours Phosphate Butanol Sodium 175° C. 36 hoursBenzoate Butanol Potassium 175° C. 36 hours Thiocyanate Butanol Borax175° C. 36 hours

Likewise, Baltes et al., (U.S. Pat. No. 3,162,658) disclose the use ofnonaqueous solvents and various metallic bases as catalysts for theconjugation of fatty acids. The various reaction parameters of themethods described by Baltes et al. are summarized in Table 2. Baltes etal. also disclose the use various low boiling point solvents. As most ofthese reactions were conducted at temperatures above the boiling pointof the solvent employed, it is apparent that the reactions wereconducted under pressure, which is an independent factor influencing theformation of octadecadienoic acid isomers. The product derived fromthese reactions will thus contain undesirable isomers.

TABLE 2 Patent 3,162,658 Solvent Catalyst Temperature Time Methanol KOH60-140° C. variable Methanol Potassium 140° C. variable MethylateButanol Potassium 140° C. variable Methylate Ethanol Potassium 140° C.variable Methylate Isopropanol Potassium 120-140° C. variable MethylateHeptane/ Potassium reflux variable 3° Butanol Butylate 3° Butanol Cesium140° C. variable Butylate Ethylene Potassium 140-160° C. variableDiamine Methylate Methanol Sodium 140° C. variable Amide

II. Controlled Isomerization Reactions

The CLA of the present invention lacks significant amounts of isomerssuch as the 8,10 isomer, the 11,13 isomer, and the various trans-transisomers. These compositions were produced by a tightly controllednonaqueous alkali isomerization process presented in flow diagram formin FIG. 1 and by isomerization with alkali alcoholate cataylsts. In someembodiments, sunflower oil, safflower oil, or corn oil are reacted at anambient pressure under an inert gas atmosphere with an excess of alkaliin a high-boiling point solvent, namely propylene glycol at atemperature below the boiling point of the solvent. In otherembodiments, sunflower oil, safflower oil, or corn oil are reacted inthe presence of an alkali alcoholate catalyst and a small amount of asuitable solvent.

A. Sources of Linoleic Acid

The preferred oils for conjugation are sunflower, safflower oil, andcorn oil. As compared to soybean oil, these oils have lowerconcentrations of undesirable components such as phosphatides andsterols. These undesirable components may contribute to the formation ofgums which foul the conjugation equipment and other undesirablepolymers. Various properties of these oils are summarized in Tables 3,4, and 5.

TABLE 3 COMPARISON OF CONTAMINANTS Phosphatides Soybean  1.5-3.0%Sunflower .4-1% Sunflower .4-1%

TABLE 4 Sterols (unsaponifiables by percent*) Soybean SunflowerSafflower Campesterol 20* Campesterol 8 Campesterol 13 Stigmasterol 20Stigmasterol 8 Stigmasterol 9 β-Sitosterol 53 β-Sitosterol 60β-Sitosterol 52 Δ⁵ Avensterol 3 Δ⁵ Avensterol 4 Δ⁵ Avensterol 1 Δ⁷Stigmasterol 3 Δ⁷ Stigmasterol 15 Δ⁷ Stigmasterol 15 Δ⁷ Avenasterol 1Avenasterol 4 Avenasterol 3 0.36% total in oil 0.36% total in oil 0.36%total in oil *May not equal 100

TABLE 5 Soybean Sunflower Safflower Iodine Value 134.6 135.4 143.6Saponification 190.7 190.6 190.3 value Unsaponification value .6 .7 .6

B. Isomerization with Propylene Glycol as a Solvent

In some embodiments of the present invention, the conjugated linoleicacid is produced by nonaqueous alkali isomerization. The reactionconditions of the controlled isomerization process allow for precisecontrol of the temperature (and constant ambient pressure) of theconjugation process. Preferably the alkali is an inorganic alkali suchas potassium hydroxide, cesium hydroxide, cesium carbonate or an organicalkali such as tetraethyl ammonium hydroxide. The catalyst is preferablyprovided in a molar excess as compared to the fatty acid content of oil.The solvent is propylene glycol. Preferably, the reaction is conductedwithin a temperature range 130 to 165° C., most preferably at about 150°C. The time of the reaction may vary, however, there is an increasedlikelihood of the formation of undesirable isomers when the reaction isconducted for long periods of time. A relatively short reaction time of2.0 to 6.5 hours has proved satisfactory for excellent yields.

It will be understood to a person skilled in the art that to produce thedesired composition, the reaction conditions described above may bevaried depending upon the oil to be conjugated, the source of alkali,and equipment. Preanalysis of a particular oil may indicate that theconditions must be varied to obtain the desired composition. Therefore,the temperature range, pressure, and other reaction parameters representa starting point for design of the individual process and are intendedas a guide only. For example, it is not implied that the describedtemperature range is the only range which may be used. The essentialaspect is to provide precise temperature control. However, care must betaken because increasing the pressure may lead to less than completeisomerization and the formation of undesirable isomers. Finally, thelength of the conjugation reaction may be varied. Generally, increasingamounts of undesirable isomers are formed with increasing length orreaction time. Therefore, the optimal reaction time allows the reactionto go nearly or essentially to completion but does not result in theformation of undesirable isomers.

Following the conjugation reaction, the resulting CLA containingcomposition may be further purified according to FIG. 1. To separate thefatty acids from the conjugation reaction mix, the reaction mix iscooled to approximately 95° C., an excess of water at 50° C. is added,and the mixture slowly stirred while the temperature is reduced to about50° C. to 60° C. Upon addition of the water, a soap of the fatty acidsis formed and glycerol is formed as a by-product. Next, a molar excessof concentrated HCl is added while stirring. The aqueous and nonaqueouslayers are then allowed to separate at about 80-90° C. The bottom layercontaining water and propylene glycol is then drawn off. The remainingpropylene glycol is removed by vacuum dehydration at 60-80° C.

The dried CLA composition may then preferably be degassed in degassingunit with a cold trap to remove any residual propylene glycol. Next, theCLA is distilled at 190° C. in a molecular distillation plant at avacuum of 10⁻¹ to 10⁻² millibar. The advantage of this purificationsystem is the short time (less than one minute) at which the CLA is heldat an elevated temperature. Conventional batch distillation proceduresare to be strictly avoided since they involve an elevated temperature ofapproximately 180-200° C. for up to several hours. At these elevatedtemperatures the formation of undesirable trans-trans isomers willoccur. Approximately 90% of the feed material is recovered as a slightlyyellow distillate. The CLA may then be deodorized by heating to about120-170° C., preferably at about 150° C. for 2 hours to improve smelland taste. Excessive heat may result in the formation of trans-transisomers. These procedures produce a CLA composition with a solvent levelof less than about 5 ppm, preferably less than about 1 ppm. This processeliminates toxic trace levels of solvent so that the resultingcomposition is essentially free of toxic solvent residues.

The processes described above are readily adaptable to both pilot andcommercial scales. For example, 400 kg of safflower oil may beconjugated at 150° C. for 5 hours in 400 kg of propylene glycol with 200kg KOH added as a catalyst. The resulting CLA may then be purified asdescribed above. Further, commercial scale batch systems may be easilymodified to produce the desired CLA composition. For example, stainlesssteel reactors should be preferably glass lined to prevent corrosion dueto pH levels of below 3.0. However, it should be noted that conjugationprocesses utilizing nonaqueous solvents are generally less corrosivethan those conducted with water.

Several comparative experiments were carried out to highlight the keyproperties of the present CLA compositions in contrast to those madeunder either suboptimal conditions or in accordance with the aqueousalkali methods of the prior art. In Example 1, the CLA was prepared bythe present method. CLA was produced by the conventional aqueous alkalimethod in Example 2. In Example 3, the reaction of Example 1 issubstantially repeated, only at high temperature. Finally, in Example 4,the aqueous alkali reaction substantially identical to that of Example 2is run at low temperature. The precise conditions and details of eachexperiments are set forth in the Examples. The profiles of the analysisof the CLA isomer content are set forth in Tables 6-11.

Referring to the data in Table 6, the relative area percentage is givenfor each identified peak corresponding to the individual isomers, foreach of the four experiments. The GC plot gave a number of peaks foreach sample tested. The area under each of these peaks was integrated toobtain a total value. The identity of the peak was determined by itsrelative position, from published atlases of standard elution profiles,and the scientific literature. The top row represents the residual valuefor unconjugated starting material, 9,12-linoleic acid. Both low andhigh temperature reaction in propylene glycol gave extremely highconversions of over 99 percent of the total starting material.

Referring to column 1, it is apparent that unlike any of the controlcompositions, in Example 1, a peak corresponding to 11,13 mixture ofisomers, the peak corresponding to c11,c13 specifically, the peaks forany of the 8,10 isomers, and the peak for unidentified isomers are allentirely missing. In the case of c9,t11 isomer, the peaks in GC for boththe 8,10 and 9,11 isomers are superimposed, and are here resolved onlyfor Example 1 material by subtracting out that portion of the peakidentified as 8,10 by NMR studies. This was not done in the otherexperiments, so that row 3 gives the values for combined 8,10 and 9,11for Examples 2-4. In general, for the 8,10, 11,13, and unidentifiedisomers, a value of less than 1 percent down to undetectable is oftherapeutic and nutritional value, because it reduces to trace levelspotentially deleterious contaminants, especially those known to havesuspect absorption pathways in lipogenesis. In non-ruminants, forexample, addition of 0.25 to 2.5 percent CLA to the diet can increasethe incidence of CLA in tissues to approximate that in ruminants, sothat other animals can be a source of CLA provided adulterating isomersare not present.

Example 2 provides a typical aqueous alkali product representative ofconventionally manufactured CLAs. Conversion is less efficient bothoverall, and in producing the c9,t11 and t10,c12 isomers. Note also ahigh percentage of the suspect 11,13 isomers, and a significantpercentage of unidentified material.

Example 3 illustrates the criticality of the temperature parameter. Anupward shift in temperature in propylene glycol media sharply increasesthe amount of the contaminating isomers at the expense of the c9,t11 andt10,c12 isomers. Also of interest, at the higher temperature there is adramatic increase in the trans, trans species, as double bondrearrangements are favored which yield a more stable electronconfiguration at levels of increased energy stress.

Example 4 illustrates that decreasing the temperature in the aqueousalkali system, in fact, reduces the amounts of some of the contaminatingisomers. However, there is a dramatic drop in yield, and the level ofthe 11,13 group of isomers remains very high, suggesting that theformation of this electron configuration is influenced more by theaction of base in an aqueous medium, than is explained by overallkinetic energy in the system. Note also the extremely long reaction timeof 22.5 hours; too long for an efficient industrial scale batch process.

Table 6 converts the relative isomer percentages in the variousreactions as a function of peak area to their corresponding peak ratios.The present process produces a virtually complete conversion of9,12-linoleic acid to an approximate equal amount of each of the twodesired CLA isomers. At the higher temperature, even in propyleneglycol, the incidence of the 11,13 isomer is still less one third thatof the low temperature aqueous alkali process.

C. Isomerization with Alcoholate Catalysts

In some embodiments, the present invention also provides methods forproducing alkyl esters of CLA. After fat splitting and dehydration, thefree fatty acids are combined with methanol or another monohydric lowmolecular weight alcohol and heated to the temperature at which thealcohol boils. Esterification proceeds under refluxing conditions withremoval of the reaction water through a condenser. After the addition ofa further quantity of the same or a different monohydric alcohol analcoholate catalyst is blended into the ester mix (See, e.g., U.S. Pat.No. 3,162,658, incorporated herein by reference). Typical alcoholatecatalysts are sodium or potassium ethoxide, or their methyl, butyl, orpropyl counterparts.

In the esterification, methanol or ethanol are preferred, although otherbranched or straight chain monohydric alcohols may be used. The longerthe aliphatic chain of the alkyl group, the more lipid compatible thematerial becomes. Also the viscosity tends to increase. For differenttypes of feed or food, whose consistency varies, product of varyingviscosity can be used to obtain the desired flow or compoundingcharacteristics without affecting the therapeutic or nutritionalproperties arising from the CLA moieties. The theory and practice ofesterification are conventional. A basic explanation of the most commonmethods is set forth in the McCraw-Hill Encyclopedia of Science &Technology, McGraw-Hill Book Co., N.Y.: 1996 (5th ed.). The animal andhuman body has a variety of esterases, so that the CLA-ester is cleavedto release the free fatty acids readily. Tissue uptake may have adifferent kinetics depending on the tissue involved and the benefitsought.

In the isomerization step, it was found that alcoholate catalysisproduced a much superior product than aqueous alkali mediatedisomerization. The latter process always produced undesirable isomerseven under mild reaction conditions. The milder conditions do give loweramounts of unwanted isomers, but at the great expense of yield, as shownin the Examples. In most systems the appearance of the c9,t11 andt10,c12 isomers dominates and they are formed in roughly equimolaramounts. It has not heretofore been possible to control theisomerization of the one isomer to the exclusion of the other. While itis desirable to increase the percentage of one or the other isomer(depending on the physiological effect to be achieved), at present thismust largely be carried out by adding an enriched source of the desiredisomer.

The preferred starting materials for conjugation with alcoholatecatalysts are sunflower oil, safflower oil, and corn oil. Each of theseoils contains high levels of linoleic acid and low levels of linolenicacid. As shown in Example 18, conjugation of linolenic acid results inthe formation of several uncharacterized fatty acid moieties, thebiological properties of which are unknown. Previous conjugationprocesses were not concerned with the production of unknown compoundsbecause the products were used in drying oils, paints and varnishes andnot in products destined from human or animal consumption. Accordingly,the CLA produced by those processes with oils containing high levels oflinolenic acid were not suitable for nutritional uses.

In some embodiments, it is further contemplated that glycerol and estersof glycerol should be removed before making monoesters of fatty acids.Traces of glycerol present during conjugation contribute to theproduction of trimethoxypropane and triethoxypropane. Therefore, priorto conjugation, it is preferable to distill monoesters obtained byalcoholysis.

D. Triacylglyceride Derivatives of CLA

The present invention contemplates the use of derivatives of CLA. Forexample, CLA may be free or bound through ester linkages as describedabove or provided in the form of an oil containing CLA triglycerides, asdescribed in Examples 5, 6, and 14. In these embodiments, thetriglycerides may be partially or wholly comprised of CLA attached to aglycerol backbone. The CLA may also preferably be provided as amethylester or ethylester as described in Examples 8 and 9. Furthermore,the CLA may be in the form of a non-toxic salt, such as a potassium orsodium salt (e.g., a salt formed by reacting chemically equivalentamounts of the free acids with an alkali hydroxide at a pH of about 8 to9).

In one embodiment of the present invention, a novel triacylglycerol issynthesized comprising the novel CLA isomer mixture disclosedhereinafter for non-aqueous isomerization of linoleic acid fromsunflower and/or safflower oils. The pure triacylglycerols highlyenriched for CLA (90-96 percent) may be confirmed by H NMR.Esterification proceeds using immobilized Candida antarctica Lipase.Preferably, the CLA will contain at least 40 and upwardly 45-48 percentof c9,t11-octadecadienoic and t10,c12-octadecadienoic acids, andmixtures thereof. There will be less than one percent esters 8,10;11,13; and trans, trans isomers or less than five percent in theaggregate. In some embodiments, the resultant triacylglycerol is notpurified further to remove all levels of phosphatidyl and sterolresidues. But those levels remaining from isomerization of sunflower andsafflower oils will be adequate for commercial applications involvingsafe, edible products in feed and food. In other embodiments, thetriacylglycerol is further purified by molecular distillation.

The immobilized Candida antarctica lipase is to be employed in a mannersimilar to that described for n-3 type polyunsaturated fatty acids, inHarraldson et al. The esterification reaction is conducted at 50°-75°C., preferably 65° C., in the absence of any solvent and a vacuumemployed in order to remove the co-produced water or alcohols (fromesters) upon formation. This shifts the triacylglycerol production tocompletion and ensures a highly pure product virtually free of any mono-and diacylglycerols in essentially quantitative yields. Stoichiometricamounts of free fatty acids may be used, i.e. 3 molar equivalents asbased on glycerol or 1 molar equivalent as based on number of molequivalents of hydroxyl groups present in the glycerol moiety. Only 10%dosage of lipase as based on total weight of substrates is needed, whichcan be used a number of times. This is very important from theproductivity point of view. All this, together with the fact that nosolvent is required, renders this process a high feasibility from thescaling-up and industrialization point of view, since the cut in volumeand bulkiness is enormous. Also, a slight excess (<5/5) of free fattyacids may be used in order to speed up the reaction toward the end andensure a completion of the reaction.

At the initiation of the reaction, the 1- or 3-mono-acyglyeride isformed first, followed by the 1,3 diacylglyeride, and finally thetriglyceride at the more extended reaction times. The mono- anddiacylglyerides are useful intermediates in that they manifestbiological activity, but have greater solubility in aqueous cellularenvironments and can participate in alternative molecular syntheticpathways such as synthesis of phospholipids or other functional lipids.In contrast, triglycerides are frequently deposited intact in cellmembranes or storage vesicles. Thus, the administration of CLA in mono-,di- or triglycerol form rather than free fatty acid or ester, mayinfluence the mode and distribution of uptake, metabolic rate andstructural or physiological role of the CLA component.

III. Stabilization of CLA Compounds

The present invention also contemplates stabilization of CLA containingcompounds, including but not limited to, CLA, esters of CLA, andtriglycerides of CLA by preventing oxidation of the compounds. Thepresent invention is not limited to any one mechanism. Indeed, anunderstanding of the mechanism of the invention is not necessary toproduce the composition or perform the methods of the present invention.Nevertheless, unlike non-conjugated fatty acids, CLA does not appear toform peroxide breakdown products. This was demonstrated experimentallyby measuring peroxide values (PV) spectrophotometrically by achlorimetirc ferric thiocyanate method. After storage in open glass, thePV of CLA was 32; in comparison, the value for linoleic acid was 370.

CLA forms volatile organic compounds during breakdown, including hexane.Products stored in a steel drum for several weeks were found to containup to 25 ppm hexane. Hexane has a characteristic taste and smell that isundesirable in food products. Oxidation of CLA appears to be caused bythe presence of metal contaminants. Thus, a system for removal of suchcompounds that promote oxidation during purification is advantageous.

Furthermore, it is also advantageous to add compounds to CLApreparations to decrease oxidation during storage. Compounds thatprevent oxidation (antioxidants) have two general mechanisms of action.The first is the prevention of oxidation by lipid peroxide radicalscavenging. Examples include but are not limited to tocopherols andascorbylpalmitate. The second mechanism for preventing oxidation is bythe chelation of metal ions. Examples of metal oxidant chelatorsinclude, but are not limited to, citric acid esters and lecithin. Somecommercially available compounds (e.g., Controx, Grumau (Henkel),Illertissen, Del.) include both peroxide scavengers and metal chelators(e.g., lecithin, tocopherols, ascorbylpalmitate, and citric acidesters). In some embodiment of the present invention, metal oxidantchelators are added to CLA containing compounds to prevent oxidation. Inother embodiments, a combination of metal oxidant chelators and peroxidescavengers is included in the CLA composition.

In some embodiments, gas chromatography/mass spectroscopy is used indetect the presence of volatile organic breakdown products of CLA. Inother embodiments, oil stability index (OSI) measurements are used todetect the presence of volatile organic breakdown products of CLA. Insome embodiments of the present invention methods for the removal ofpro-oxidants (e.g., iron) from CLA samples are provided. Methodsinclude, but are not limited to distillation or by adsorption. In someembodiments of the present invention, compounds are added to preventoxidation of CLA.

Example 12 illustrates the measurement of volatile organic compounds bygas chromatography followed by mass spectroscopy. CLA is prepared by themethod of Example 11. GC/MS is performed and peaks are identified byusing reference materials (e.g., Wiley reference search). Table 28 liststhe compounds identified and their relative abundance. Volatile organiccompounds identified include pentane, hexane, heptane, 2-butenal,ethanol, 3-methyl butanal, 4-methyl pentanone, hexanal, heptanal,2-pentyl furan, and octanal. Example 12 illustrates that samples of CLAcontain undesirable volatile organic compounds. It is understood by oneskilled in the art that samples may contain additional volatile organiccompounds, depending on the starting materials and the exact reactionconditions.

In one illustrative example, it is demonstrated that the production ofvolatile organic acids increases over time. Example 13 shows therelative amounts of pentane and hexane in a CLA solution before andafter storage in open air at 60° C. for 21 days. The results are shownin Table 28. The amount of both pentane and hexane increased byapproximately two fold after 21 days. This example illustrates that thelevel of volatile organic compounds present as oxidation products of CLAincreases over time.

In preferred embodiments, precautions are taken during purification toprevent oxidation during storage. These precautions include the removalof compounds that serve as pro-oxidants, including but not limited toiron or other metals. In some embodiments, metals are removed bytreating with adsorbing agents, including but not limited to bleachingearth, active charcoal zeolites, and silica. In other embodiments, thepro-oxidants are removed by distillation.

Example 16 provides an illustrative example of one method for adsorbingmetals. In this example, silica is used as the adsorbing agent. Atriacylglyceride prepared by the method of example 14 was firstdeodorized at an elevated temperature and pressure. The sample was thenmixed with silica and heated under vacuum. The present invention is notintended to be limited to the adsorption conditions described in Example16; other methods of adsorption known to those skilled in the art arecontemplated.

In some embodiments, pro-oxidants are removed in a distillation process.An illustrative example is given in Example 14. In this example,distillation of a triacylglyceride of CLA is performed on a moleculardistillation apparatus. Distillation is carried out at 150° C. and apressure of 10-2 mbar. The present invention is not intended to belimited to the conditions described for distillation. Other temperaturesand pressures are within the scope of the present invention.

In some embodiments, oxidation of CLA is prevented by the addition ofmetal oxidant chelators or peroxide scavengers to the finished product.In some embodiments, the amount of oxidation is measured by the oilstability index (OSI). The OSI (See e.g., AOCS official method Cd12b-92) is a measurement of an oil's resistance to oxidation. It isdefined mathematically as the time of maximum change of the rate ofoxidation. This rate can be determined mathematically. Experimentally,the OSI is calculated by measuring the change in conductivity ofdeionized water is which volatile organic acids (oxidation products) aredissolved. When performing OSI measurements, it is important to avoidcontamination by trace amounts of metals, which can accelerate theoxidation process. This is generally accomplished by careful washing ofall glassware used with a cleaning solution lacking chromate orsurfactants. Water must be deionized and all solvents must be of ahighly purified grade.

An example illustrating OSI measurement of CLA in the presence orabsence of antioxidants is given in Example 15. In Example 15, atriacyglyceride of CLA is prepared by the method of Example 14. Samplesare placed in open dishes with varying amounts (0-0.1%) of fourantioxidants (Controx Grunau (Henkel), Illertissen, Del.), Herbalox (anextract of rosemary; Kalsec, Kalamazoo, Mich.), Covi-OX (Grunau(Henkel), Illertissen, Del.), and alpha-tocopherol). The OSI iscalculated as described above. Results are shown in Table 29 and FIG. 2.The addition of alpha-tocopherol did not significantly increase the OSIvalue. Herbalox increased the value by approximately 2-3 fold. Covi-OXand Controx increased the OSI values by a greater amount, approximately4 and 6 fold, respectively. This experiment demonstrated that theaddition of antioxidants can slow the oxidation of CLA containingcompounds during storage.

IV. Administration of CLA Containing Compounds

The conjugated linoleic moieties of the present invention may beprovided in a variety of forms. In some embodiments, administration isoral. The CLA moieties may be formulated with suitable carriers such asstarch, sucrose or lactose in tablets, pills, dragees, capsules,solutions, liquids, slurries, suspensions and emulsions. Preferably, theCLA formulations contain antioxidants, including, but not limited toControx, Covi-OX, lecithin, and oil soluble forms of vitamin C (ascorbylpalmitate). The CLA may be provided in aqueous solution, oily solution,or in any of the other forms discussed above. The tablet or capsule ofthe present invention may be coated with an enteric coating whichdissolves at a pH of about 6.0 to 7.0. A suitable enteric coating whichdissolves in the small intestine but not in the stomach is celluloseacetate phthalate. In some embodiments, the CLA is provided as softgelatin capsules containing 750 mg 80% CLA (Tonalin™). The CLA may alsobe provided by any of a number of other routes, including, but notlimited to, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual or rectalmeans. Further details on techniques for formulation for andadministration and administration may be found in the latest edition ofRemington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

An effective amount of CLA moiety may also be provided as a supplementin various food products, including animal feeds, and drinks. For thepurposes of this application, food products containing CLA means anynatural, processed, diet or non-diet food product to which exogenous CLAhas been added. The CLA may be added in the form of free fatty acids,esters of conjugated linoleic acid, or as an oil containing partial orwhole triglycerides of CLA. Therefore, CLA may be directly incorporatedinto various prepared food products, including, but not limited to dietdrinks, diet bars, supplements, prepared frozen meals, candy, snackproducts (e.g., chips), prepared meat products, milk, cheese, yogurt andany other fat or oil containing foods. Food products formulated withalkyl esters or conjugated linoleic acid moieties produced by alkalialcoholate catalysts contain alcohols (e.g., methyl or ethyl alcohol)depending on the solvents and cataylsts utilized. Generally, thealcohols will be present at about 1 to 10 ppm.

Furthermore, as shown above and in the Examples, CLA compositions cancontain levels of volatile organic compounds that cause the taste andsmell of food products containing the CLA to be adversely effected. Itis contemplated that the food products of the present invention thatcontain CLA compositions having less than 100 ppm volatile organiccompounds, and preferably less than 5 ppm volatile organic compounds,are superior in taste and smell to food products containing higherlevels of volatile organic compounds and will be preferred in blindtaste and smell tests. Accordingly, some embodiments of the presentinvention provide a food product containing a conjugated linoleic acidmoiety, wherein the conjugated linoleic acid moiety has a sufficientlylow volatile organic acid compound concentration so that taste and smellof the food product is not affected.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μM (micromolar); kg(kilograms); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); L or l (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); nm (nanometers); ° C. (degreescentigrade); KOH (potassium hydroxide); HCL (hydrochloric acid); Hg(mercury).

EXAMPLE 1 Isomerization of Safflower Oil Using Propylene Glycol at LowTemperature

Safflower oil was isomerized in propylene glycol at low temperaturesusing KOH as a catalyst. The isomerization apparatus consisted of atwo-necked flask with a thermometer placed in one neck, leaving a smallopening to release excess pressure. A nitrogen supply was attached tothe other neck of the flask. Solutions added to the flask were agitatedby the use of a magnetic bar and a magnetic stirrer. The temperature ofthe flask was controlled by placing the flask in a thermostat-controlledoil bath placed on the magnetic stirrer.

The flask was filled with 60.27 g propylene glycol and 28.20 g KOH andimmersed into the oil bath. The temperature was increased to 130° C. todissolve the KOH. After the KOH had dissolved, 60.09 g of safflower oilwas introduced into the flask. A high volume of nitrogen was circulatedthrough the two-neck flask for 5 min. and then reduced to a lowervolume. The mixture was heated to 150° C., which took approximately 40min. The mixture was then allowed to react at 150° C. for 3.5 hours. Atintervals, 3 ml samples were withdrawn for analysis.

The samples were placed directly into 6 ml of hot water and citric acidwas added in excess until the free fatty acids separated out as the toplayer. Heating was necessary to prevent solidification while the citricacid was added. To convert the free fatty acids into methylesters foranalysis by Gas Chromatography, 0.025 g of the free fatty acids, 5 ml ofa 4% solution of HCl and ethanol were added to a test tube. Nitrogen wasadded to the tube, then the tube was sealed and placed in a water bathat 60° C. for 20 min. The tube was then cooled and 1 ml purified waterand 5 ml isooctane were added. Nitrogen was added to the tube and thetube was shaken for 30 seconds. The resulting upper layer was added to 1μl of purified water in a new test tube and again shaken under nitrogen.The resulting upper layer was then washed of isooctane and decanted intoa third test tube. A small amount of sodium sulfate was added for waterabsorption. A 1 μl sample was then injected directly into the Gaschromatograph.

The gas chromatography conditions were as follows:

System: Perkins-Elmer Auto System Injector: Splitless at 240° C.Detector: Flame Ionization Detector at 280° C. Carrier: Helium Column:WCOT Fused Silica 0.25 mm X100M, CP-SL 88 for FAME, DF 0.2 Oven Program:80° C. (0 min.) increasing to 220° C. at 10° C. per min. and held at220° C. for 10 min.

All results are expressed as the relative peak area percentage.Standards are generally unavailable, so the peaks which eluted wereverified with other systems. GC-MS determines the number, but not theposition of cis and trans bonds. Therefore, NMR analysis was used toverify the bond positions. The main peaks were c9,t11 and t10,c12. ForNMR analysis of CLA isomers, please see Marcel S. F. Lie Ken Jie and J.Mustafa, Lipids, 32 (10) 1019-34 (1997), incorporated herein byreference.

This data, presented in Table 6 and summarized in Table 10, demonstratesthat isomerization of safflower oil using polypropylene glycol as asolvent, KOH as a catalyst, and low temperatures results in theproduction of conjugated linoleic acid lacking 8,10 and 11,13 isomers.The highly polar columns utilized in this experiment may be successfullyused to separate the 8,10 and 11,13 isomers from c9,t11 and t10,c12isomers. The 8,10 isomers tend to coelute or elute just after the c9,t11isomer. The 11,13 isomer elutes in front of the t10,c11 isomer orcoelutes with the t10,c12 isomer, depending on the column conditions.

The conjugated linoleic acid produced according to this method bycharacterized by comparing the various isomers produced. First, theisomerization reaction went essentially to completion. The completenessof the reaction is obtained by dividing the total peak area the forlinoleic acid isomers minus residual c9, t12 linoleic acid by the totalpeak area. This value is 0.994. Second, the ratio of c9,t11 and t10,c12isomers to total peak area may be determined. This value is 0.953.Third, the ratio of the t9,t11 and t10,t12 isomers to the c9,t11 andt10,c12 isomers may be determined. This value is 0.010. Fourth, theratio of the t9,t11 and t10,t12 isomers to total peak area may bedetermined. This value is 0.009. Fifth, the ratio of the t10,c12 isomerto the c9,t11 isomer may be determined. This value is 1.018. Theseratios are summarized in Table 11.

EXAMPLE 2 Aqueous Isomerization at High Temperature and Pressure

Fifty grams of water and 25.32 g NaOH were added to a high pressurereactor (Parr Model 450 ML Benchtop Alloy 400, equipped with a pressuregauge and stirrer.) The NaOH was allowed to dissolve and 94.0 gsafflower oil was added to the reactor. The reactor was closed andflushed for 2 min. with nitrogen and then all valves were closed. Thereactor was heated in an electrical gasket to 210° C. and maintained atthat temperature for 6 hours. The temperature was then reduced to 60° C.before pressure was released and the reactor opened. Two grams of theresulting solidified soap were taken from the reactor and dissolved inwater at approximately 40° C. Citric acid was then added to reduce thepH of the solution to below 6. A sample was withdrawn from the fattyacid top layer and prepared for Gas Chromatography as in Example 1.

The results of the gas chromatography are presented in Table 7 andsummarized in Table 10. These data indicate that this isomerizationmethod results in the formation of relatively high amounts of the 8,10and 11,13 isomers. Ratios are presented in Table 11.

EXAMPLE 3 Non-aqueous Alkali Isomerization of Safflower Oil at HighTemperature and Pressure

100.48 g propylene glycol and 46.75 g of KOH were added to ahigh-pressure reactor as described in Example 2. The reactor was thenheated to 130° C. to dissolve the KOH. 100.12 g of safflower oil werethen added to the KOH-propylene glycol mixture. The reactor was closed,flushed for 1 min. with nitrogen, and all valves closed. The reactor wasthen heated to 210° C. and maintained at that temperature for 1 hour.The reactor was cooled and the contents decanted into 120 g of hotwater. While stirring, 35.3 g 37% HCl and 27.59 g citric acid wereserially added to the fatty acids. A sample was taken from the top layerand dried in a vacuum flask at 60° C. A sample of the resulting fattyacids was analyzed by gas chromatography as described in Example 1.

The results are presented in Table 8 and summarized in Table 10. Thisexperiment demonstrates that isomerization of safflower oil with KOH anda non-aqueous solvent at high temperature results in the formation ofsignificant amounts of 8,10 and 11,13 isomers, as well as t9,t11 andt10,t12 isomers. Ratios are presented in Table 11.

EXAMPLE 4 Aqueous Alkali Reaction at Low Temperature

49.94 g water and 39.96 g NaOH were added to a high-pressure reactor asdescribed in Example 3. This mixture was heated until the NaOHdissolved. Next, 100.54 g of safflower oil was added to thehigh-pressure reactor, the reactor was flushed with nitrogen, and allvalves closed. The high-pressure reactor was heated to 179° C. for 22.5hours. Samples were prepared for Gas Chromatography as in Example 3. Thedata is provided in Table 9 and summarized in Table 10. This experimentdemonstrates that when low temperatures are used for aqueous alkaliisomerization, the conjugation reaction does not go to completion.Furthermore, significant amounts of the 8,10 and 11,13 isomers areproduced. Ratios are presented in Table 11.

TABLE 6 Peak Time Component Area Area Height # (Min) Name (%) (μV · s)(μV) 1 38.164 0.08 4101.65 622.28 2 49.539 C16:0 6.29 335897.80 32745.953 53.107 C16:1 0.06 3240.60 447.82 4 61.620 C18:0 2.38 127182.3012999.14 5 64.821 C18:1 c9 12.34 659111.72 52209.40 6 65.254 0.5730402.68 3475.09 7 67.263 0.11 5757.35 758.08 8 67.940 0.10 5523.00700.44 9 68.755 0.24 12816.90 1543.27 10 69.310 0.22 11803.80 1430.59 1169.846 C18:2 c9, c12 0.44 23336.75 2500.24 12 73.618 0.28 14828.701838.66 13 76.621 0.16 8400.65 1050.19 14 77.388 CLA c9, t11 36.511950669.98 124313.83 15 78.370 CLA t10, c12 37.16 1985488.96 132265.3316 78.664 CLA c9, c11 1.06 56583.10 5699.43 17 78.880 CLA c10, c12 1.2667503.55 4572.65 18 80.102 CLA t9, t11/ 0.73 39110.00 4743.28 t10, t1219 85.165 0.03 1621.65 231.32 100.00 5343381.15 384147.01

TABLE 7 Peak Time Component Area Area Height # (Min) Name (%) (μV · s)(μV) 1 36.554 0.09 4122.05 627.02 2 47.785 C16:0 6.68 290571.30 28224.343 51.280 C16:1 0.07 3188.05 425.57 4 59.787 C18:0 2.63 114362.9512678.63 5 62.923 C18:1 c9 13.12 570712.08 42259.71 6 63.346 0.7231329.22 3774.35 7 65.355 0.54 23620.70 2848.31 8 66.034 0.67 28980.783333.95 9 66.574 0.10 4370.91 594.22 10 66.811 0.35 15045.61 1469.30 1167.352 0.41 18002.20 2035.53 12 67.889 C18:2 c9, c12 1.43 62002.156714.22 13 69.200 0.09 3840.85 474.10 474.10 14 71.680 0.30 13099.101744.21 15 74.640 1.62 70689.87 4117.23 16 75.310 CLA c9, t11/ 24.871082087.96 57619.24 8, 10 17 76.032 CLA 11, 13 14.72 640440.14 42975.8618 76.277 CLA t10, c12 16.00 695923.85 63512.81 19 76.450 CLA c8, c101.26 54676.10 7614.29 20 76.626 CLA c9, c11 2.08 90411.44 10891.36 2176.881 CLA c10, c12 3.00 130593.96 11727.80 22 77.022 CLA c11, c13 1.7777065.69 9906.74 23 77.477 0.66 28867.85 3322.69 24 77.868 0.63 27391.942934.68 25 78.173 CLA 6.00 260985.40 26124.10 t9, t11/t10, t12 26 83.1400.12 5164.40 586.21 27 85.878 0.06 2735.80 347.01 100.00 4350282.35348883.46

TABLE 8 Peak Time Component Area Area Height # (Min) Name (%) (μV · s)(μV) 1 38.249 0.08 3999.70 599.26 2 49.639 C16:0 6.41 333807.80 32279.133 53.218 C16:1 0.06 3123.00 427.39 4 55.508 0.03 1322.20 190.60 5 61.753C18:0 2.55 132854.50 14939.09 6 64.104 C18:1 c9 0.03 1640.30 245.73 764.950 12.92 672672.91 53345.47 8 65.382 0.64 33297.29 3728.28 9 65.7830.03 1411.20 219.76 10 67.403 0.62 32194.66 2836.09 11 67.793 0.2412660.05 1495.10 12 68.088 0.68 35371.43 3210.82 13 68.421 0.07 3684.10473.77 14 68.635 0.04 1948.63 257.65 15 68.890 0.29 14979.18 1499.63 1669.192 0.04 2268.69 324.39 17 69.430 0.25 13028.21 1369.93 18 69.947C18:2 c9, c12 0.23 11895.70 1125.77 19 70.341 0.02 1168.20 196.75 2073.741 0.31 15930.60 1965.82 21 75.448 0.08 3906.00 387.98 22 76.7681.79 93172.74 6637.34 23 77.002 0.63 32882.76 5024.06 24 77.389 CLA c9,t11/ 15.62 813447.45 57234.62 8, 10 25 77.735 1.92 99754.50 8641.88 2678.045 CLA 11, 13 4.03 209728.35 19826.20 27 78.335 CLA t10, c12 12.63657681.44 62016.93 28 78.566 CLA c8, c10 0.64 33432.80 5277.06 29 78.727CLA c9, c11 2.21 114935.49 10791.54 30 79.079 CLA c10, c12 3.98207339.28 12766.61 31 79.663 CLA c11, c13 1.40 73036.34 6275.58 3280.516 CLA t9, t11/ 29.39 1529956.09 100323.85 t10, t12 33 82.318 0.031563.70 230.42 34 85.289 0.07 3657.50 423.53 35 88.093 0.05 2368.50301.03 100.00 5206121.30 416889.05

TABLE 9 Peak Time Component Area Area Height # (Min) Name (%) (μV · s)(μV) 1 38.154 0.09 3371.70 501.86 2 49.501 C16:0 6.80 253221.00 25807.113 53.100 C16:1 0.07 2723.55 353.01 4 55.391 0.03 1078.10 142.65 5 61.618C18:0 2.68 100015.20 11002.94 6 63.990 0.03 946.40 156.50 7 64.791 C18:1c9 13.13 489016.55 38313.02 8 65.270 0.69 25645.55 2670.46 9 67.296 0.124466.65 558.35 10 67.960 0.11 4012.70 517.76 11 68.800 0.37 13840.491314.91 12 69.370 0.30 11141.11 1245.85 13 70.001 C18:2 c9, c12 20.52764287.35 62474.10319 .72 14 73.538 0.30 11075.20 1357.19 15 76.519 0.4215662.14 1154.22 16 77.231 CLA 22.45 836230.58 56972.76 c9, t11/8, 10 1777.911 CLA 11, 13 7.56 281633.54 24467.27 18 78.197 CLA t10, c12 19.77736384.86 66688.46 19 78.559 CLA c8, c10 1.21 45158.40 3837.29 20 78.787CLA c9, c11 0.87 32564.06 3409.07 21 78.953 CLA c10, c12 0.89 33053.572499.70 22 79.413 CLA c11, c13 0.12 4453.10 353.06 23 79.792 0.134936.60 436.59 24 80.052 CLA 1.13 42203.55 4550.59 t9, t11/t10, t12 2582.298 0.03 981.60 150.46 26 82.946 0.03 1107.95 151.48 27 85.135 0.103639.90 383.36 28 87.927 0.06 2212.50 254.61 100.00 3725063.90 311570.23

TABLE 10 Relative Area Percentage Isomer Example 1 Example 2 Example 3Example 4 c9, t12 0.44 1.43 0.23 20.52 c9, t11 36.51 na na na c9, t11/<0.5* 24.87 15.62 22.45 8, 10 t10, c12 37.16 16.00 12.63 19.77 c9, c111.06 2.08 2.21 0.87 c8, c10 <0.5 1.26 0.64 1.21 c10, c12 1.26 3.00 3.980.89 t9, t11/ 0.73 6.00 29.39 1.13 t10, t12 11, 13 <0.5 10.23 4.05 7.65c11, c13 <0.5 1.77 1.40 0.12 Uniden- <0.5 2.91 4.34 0.55 tified CLATotal 76.88 72.61 74.24 54.55 Total 77.32 74.04 74.47 75.07 area *-totalpercentage of 8, 10 is less than 0.5 na-value is reflected as componentof c9, t11/8, 10

TABLE 11 Example Example Example Example Isomer Ratio 1 2 3 4 Total CLATotal peak 0.994 0.981 0.997 0.727 isomer area c9, t11 − Total peak0.953 0.552* 0.379* 0.562* t10, c12 area t9, t11 − c9, t12 − 0.0100.147* 1.040* 0.027* t10, t12 t10, c12 t9, t11 − Total peak 0.009 0.0810.395 0.015 t10, t12 area Total 11, 13 Total peak na 0.223 0.073 0.102area t10, c12 c9, t11 1.018 1.554* 0.809* 0.881* *c9, t11 includes 8, 10isomer na-no 11, 13 detected

EXAMPLE 5 The Preparation of Triacylglycerols of CLA by DirectEsterification

General. H nuclear magnetic resonance spectra were recorded on a BrukerAC 250 NMR spectrometer in deuterated chloroform as a solvent. HPLCseparations were carried out by a PrepLC™ System 500A instrument fromWaters using the PrepPak® 500/Silica Cartridge column from Millipore,eluting with 10% diethyl ether in petroleum ether. Analytical GLC wasconducted on a Perkin-Elmer 8140 Gas Chromatograph according to apreviously described procedure, as described in Haraldsson, et al., ActaChem Scanned 45: 723 (1991).

The immobilized Candida antarctica lipase was provided by Novo Nordiskin Denmark as Novozyme™. It was used directly as provided in theesterification experiments. Analytical grade diethyl ether purchasedfrom Merck was used without any purification, but synthetic graden-hexane also from Merck was freshly distilled prior to use inextractions and HPLC chromatography. Glycerol (99%) was purchased fromSigma and Aldrich Chemical Company and used without furtherpurification. The CLA concentrate was provided by Natural Lipids inNorway as free fatty acids as Tonalin™. Its purity was confirmed byanalytical GLC and high-field NMR spectroscopy which revealed someglyceride impurities. The CLA concentrate was found to contain 43.3%9-cis,11-trans-linoleic acid, 44.5% 10-trans, 12-cis-linoleic acid, 5.4%of other CLA isomers, 5.6% oleic acid and 0.6% each of palmitic andstearic acid as determined by GLC at the Science Institute.

EXAMPLE 6 The Preparation of Triacylglycerols of CLA by DirectEsterification

Immobilized Candida antarctica lipase (1.25 g) was added to a mixture ofglycerol (1.22 g. 13.3 mmol) and CLA as free fatty acid (M.wt.280.3g/mol; 11.6 g, 41.5 mmol). The mixture was gently stirred on a magneticstirrer hot plate at 65° C. under continuous vacuum of 0.01-0.5 Torr.The volatile water produced during the progress of the reaction wascontinuously condensed into liquid nitrogen cooled traps. After 48 h thereaction was discontinued, n-hexane added and the enzyme separated offby filtration. The organic phase was treated with an alkaline aqueoussolution of sodium carbonate to remove excessive free fatty acids (whenrequired). The organic solvent (after drying over anhydrous magnesiumsulfate when appropriate) was removed in vacuo on a rotary evaporatorfollowed by high-vacuum treatment to afford the virtually pure productas a slightly yellowish oil (10.9 g; average M.wt. 878.6 g/mol; 93%yield). When stoichiometric amounts of free fatty acids were used,titration by standardized sodium hydroxide was applied to determine thefree fatty acid content of the crude reaction product (less than 1% freefatty acid content as based on number of mol of ester groups,corresponding to at least 99% incorporation, which is equivalent to theminimum of 97% triglyceride content). The crude product was directlyintroduced into HPCL eluting with 10% diethylether in n-hexane to afford100% pure triglyceride as a colourless oil. 250 MHz 1H NMR (CDC13) 8(ppm) 6.35-6.23 (3H, ddt, Jtrans=15.0 Hz, J=10.9 Hz,Jally1=1.3,═CHCH═CH), 5.98-5.90 (3H, dd, Icis=10.9, J=10.9, —CH═CHCH═),5.71-5.59 (3H, dtd, Jtrans=15.0 Hz, J=6.9 Hz, J=6.9 Hz, J=2.2 Hz,═CH═CHCH2-), 5.35-5.26 (4H, m, ═CH2CH═CH— and —CH2C —ICH2-), 4.33-4.26(2H, dd, Jgem=11.9 Hz, J=4.3, —CH2CHCH2-), 4.18-4.10 2H, dd, Jgem=1.8Hz, J=6.0, —CH2CHCH2-), 2.37-2.31 (6H, t, J=7.4 H2, —CH2COOR), 2.19-2.05(12H, m, —CH2CH═CH—), 1.66-1.60 (6H, qu., J=Hz, —CH2CH2COOR), 1.43-1.30(18H, m, —CH2-), 0.91-0.86 (9H, t, J=6.7 Hz, —CH3). 13C-NMR (CDC13): 8(ppm) 173.2, 172.8, 134.6, 130.0, 128.6, 125.5, 68.8, 62.0, 34.0, 32.9,31.6, 29.6-28.9 (6C), 27.6, 24.8, 22.5, 14.1.

In order to monitor the progress of the reaction and provide moredetails about the composition of individual glycerides during thereaction, samples were collected regularly as the reaction proceeded.They were analyzed by HNMR spectroscopy and provided a good insight intothe composition of mono-, di- and triacylglycerols during the progressof the reaction. The results are demonstrated in Table 12 below. As canbe noticed from the table, 1,3-diacylglycerols dominated the reactionmixture during the first two hours of the reaction. After 4 hourstriacylglycerols took over and had reached 98% composition after 22hours and 100% after 48 hours. As would be expected 1,2-diacylglycerolsreached considerably lower levels than the 1,3-diacylglycerols.1-monoacylglycerols reached a maximum during the first hour of thereaction, but 2-monoacylglycerols were not detected throughout thereaction.

TABLE 12 Residual Time % Incorporation FFA h 1-MG 1,2-DG 1,3-DG TG % 0 00 0 0 100 1 8.3 15.2 39.4 7.8 29.3 2 2.7 9.3 46.5 17.4 24.1 4 1.7 7.925.4 49.4 15.5 6 0.5 5.2 16.0 68.1 10.1 8 0.0 3.9 9.9 80.5 5.7 10 0.03.0 7.0 85.8 4.2 12 0.0 2.7 5.6 89.2 2.5 22 0.0 1.0 1.4 95.8 1.8 48 0.00.0 0.0 100 0.0

EXAMPLE 7 Effect of Varying Temperature and Reaction Duration on CLAYield and Composition

The effect of temperature and reaction duration on the conjugation ofsafflower oil was determined. Water and NaOH were added to a highpressure reactor (Parr Model 450 ML Benchtop Alloy 400, equipped with apressure gauge and stirrer) as indicated in Table 1, columns 1 and 2.The NaOH was allowed to dissolve and safflower oil (column 3) was addedto the reactor. The reactor was closed and flushed for 2 min. withnitrogen and then all valves were closed. The reactor was heated in anelectrical gasket to the desired temperature (column 4) and maintainedat that temperature for the desired time (column 5). The temperature wasthen reduced to 60° C. before pressure was released and the reactoropened. For each reaction, two grams of the resulting solidified soapwere taken from the reactor and dissolved in water at approximately 40°C. Citric acid was then added to reduce the pH of the solution to below6. A sample was withdrawn from the fatty acid top layer and prepared forGas Chromatography.

The results of the gas chromatography are presented in column 6 (totalpercentage of 9,11 and 10,12 isomers), column 7 (total percentage of11,13 isomers), and column 8 (total percentage of all CLA isomers oryield). These data indicate that as reaction duration and temperatureincrease, the total amount of conjugation and the percentage of 11,13isomers increase. Under conditions where formation of the 11,13 isomeris low, the total amount of conjugation is also low.

TABLE 13 Saf- Wa- flower Mean 9,11 + CLA ter NaOH Oil t. ° C. of Time10,12 11,13 total gram gram gram reaction hours area % area % area %50.21 29.93 99.94 189 6.36 45.99 5.73 55.86 70.20 29.93 99.94 187 6.4044.94 3.23 51.28 50.10 30.17 100.74 183 6.39 40.23 3.37 48.07 49.9129.93 100.40 179 6.52 32.00 1.48 34.92 49.97 29.80 100.02 179 10.0841.86 3.12 48.21 49.94 39.84 99.84 179 6.30 32.6 3.04 37.12 29.50 24.8399.21 240 3.25 28.37 10.78 71.58 30.33 25.15 100.43 221 2.30 40.87 14.7272.61 49.92 30.00 100.36 150 6.34 7.07 0 7.44

EXAMPLE 8 Conjugation of Safflower Fatty Acid Methylester (FAME)

The reaction was carried out in a closed vessel. The followingcomponents were mixed together: 100 g safflower FAME and a mixture ofapproximately 2.8 g KOCH₃ and 2.8 g methanol. There was probably moreKOMe than methanol due to evaporation of methanol during mixing of thetwo components. The mixture was stirred for 5 hours at 111-115 deg C. innitrogen atmosphere in a closed reaction vessel. The distribution ofisomers was analyzed by Gas Chromatography. The results are summarizedin Table 2. The raw GC data is presented in Table 3. These data indicatethat the conjugation safflower FAME may be accomplished under mildconditions, resulting in a product lacking appreciable amounts ofundesirable 8,10 and 11,13 isomers.

TABLE 14 Isomer Distribution Palmitic acid 6.6% Stearic acid 2.7% Oleicacid 12.9% Linoleic acid 5.7% (unconjugated) CLA c9,t11 34.1% CLAt10,c12 33.3% CLA c,c 1.8% CLA t,t 1.0% CLA total 70.2%

EXAMPLE 9 Large Scale Batch Production of Conjugated Safflower FAME

The production of safflower conjugated FAME may be divided into twosteps, methanolysis and conjugation. For methanolysis, 6,000 kgsafflower oil was drawn into a closed reactor. The reactor was purgedwith nitrogen at atmospheric pressure, and 1150 liters of methanol and160 kg of NaOCH₃ (30% solution) were added. The mixture is heated to 65°C. while stirring, and reacted at 65° C. for 2 hours. The resultingbottom layer was decanted while the reactor was purged with nitrogengas. 1000 liters of water (40-50° C., into which 50 kg citric acidmonohydrate has been dissolved) was then added while stirring. Thelayers were allowed to separate (approx. 60 min.) and the bottom layerdecanted while purging the reactor with nitrogen gas. The resultingsafflower FAME product was dried at 80° C. under vacuum for one hour.

To conjugate the safflower FAME, 250 kg of KOCH₃ dissolved in methanolto form a paste was added to the reactor. The mixture was then heated to120° C. while stirring and the reaction allowed to continue for 3 hours.The mixture was cooled to 100° C., and 1000 liters of water (40-50° C.,into which 50 kg citric acid monohydrate has been dissolved) was addedwhile stirring. The mixture was stirred for 15 minutes and then thelayers were allowed to separate for 20 minutes. The bottom layer wasdecanted and the product dried at 80° C. for 1 hour and then storedunder nitrogen.

The resulting CLA was analyzed using a Perkin Elmer Autosystem XL GCunder the following conditions:

Column: WCOT Fused Silica 100 m × 0.25 mm, Coating CP SIL 88 Carrier: Hegas, 30.0 PSI Temp: 220 C Run time: 35-90 min. Inject.: Splitless, 240 CDetect.: FID, 280 C

The GC results are summarized in Tables 15 and 16.

TABLE 15 Peak Time Component Area Area Height # (min) Name (%) (μVs)(μV) 1 46.874 C16:0 6.37 29874.50 4026.29 2 58.685 C18:0 2.61 12231.701542.34 3 62.141 C18:1 c9 13.14 61668.78 7369.08 4 62.652 0.70 3263.62391.92 5 66.404 0.35 1627.60 177.41 6 66.917 0.26 1239.15 157.35 767.583 C18:2 5.75 26964.95 3153.80 c9, c12 8 70.631 0.25 1171.90 141.419 75.011 CLA 34.42 161529.90 17544.79 c9, t11 10 75.936 CLA 33.48157129.82 17157.21 t10, c12 11 76.400 CLA 0.84 3935.70 302.61 c9, c11 1276.631 CLA 0.49 2316.98 279.31 c10, c12 13 77.905 CLA t, 1.35 6344.50710.88 t9, 11 + 10, 12 100.00 469299.10 52954.41

EXAMPLE 10

The following are examples of typical animal rations containing the CLAfree fatty acids, triglycerides, and esters of the present invention.

A. Pig Starter Rations

TABLE 16 Ingredients lbs. kgs. Corn, yellow (8.4% protein) 1067 484.7Soy bean meal, solvent extracted, 570 259 dehulled (47% protein) CLA 52.3 Whey, dried (12.0% protein) 300 136 Dicalcium phosphate 24 11Limestone 16 7 Iodized salt 5 2 Trace mineral premix 5 2 Vitamin premix8 4 Totals 2000 908

B. Grower-finisher Rations For Pigs (From 40-240 lbs[18-109 kgs])

TABLE 17 Ingredients lbs. kgs. Corn, yellow (8.4% protein) 1566 Soybeanmeal, solvent extracted (44% 380 protein) CLA 5 Dicalcium phosphate 21Limestone 15 Iodized Salt 5 Trace Mineral Premix 3 Vitamin Premix 3Total 2000

C. Pig Grower Finisher Rations (For Pigs 121-240 lbs[55-109 kgs])

TABLE 18 Ingredients lbs. kgs. Corn, yellow (8.4% protein) 1687 Soybeanmeal, solvent extracted (44% 265 protein) CLA 5 Dicalcium phosphate 18Limestone 15 Iodized salt 5 Trace mineral premix 2 Vitamin premix 3Total 2000

Composition and Analysis of Pig Trace Mineral Remix

TABLE 19 Element Source Amount (lbs) Copper (Co) Copper Sulfate 1.500Iodine (I) Potassium Iodide 0.010 Iron (Fe) Ferrous Sulfate 25.000Manganese (Mn) Manganese Sulfate 2.500 Selenium (Se) Sodium Selemite)0.025 Zinc (Zn) Zinc Sulfate 25.000 Carrier 45.965 Total 100.000

Composition of Pig Vitamin Premix

TABLE 20 Vitamins Amount Essential Vitamin A . . . (million IU) 5.0Vitamin D . . . (million IU) 0.6 Vitamin E . . . (thousand IU) 26.0Niacin . . . (g) 25.0 d-Pantothenic acid . . . (g) 20.0 Riboflavin . . .(g) 6.0 Vitamin B-12 . . . (mg) 25.0 Optional Biotin . . . (g) 0.3Menadione . . . (g) 4.0 Carrier . . . to 10 lbs Total 10.0

D. 18% Protein Layer Rations for Hens

TABLE 21 Ingredients lbs. kgs. Ground yellow corn 1242 564.5 CLA 5 2.3Alfalfa meal, 17% 25 11.3 Soybean meal, dehulled 451.6 205.3 Meat andbone meal (47%) 50 23.0 DL-methionine 1.0 .5 Dicalcium phosphate 7 3.1Ground limestone 174 79.1 Iodized salt 7 3.1 Stabilized yellow grease 3717.2 Mineral and vitamin supplements Calcium pantothenate (mg) 5,000Manganese (g) 52 Selenium (mg) 90.8 Zinc (g) 16 Vitamin A (IU) 6,000,000Vitamin D₃ (IU) 2,000,000 Choline (mg) 274,000 Niacin (mg) 12,000Riboflavin (mg) 2,000 Vitamin B-12 6 Total 2000 909.4

E. Starter and Finisher Rations for Broilers

TABLE 22 Starter Finisher (25 days to (up to 24 days) market)Ingredients lbs. kgs. lbs. kgs. Ground yellow corn 1,106 503 1235 561CLA-ester 5 2.3 5 2.3 Soybean meal, dehulled 605 275 420 191 Alfalfameal, 17% — — 25 11 Corn gluten meal, 60% 50 23 75 34 Fish meal,herring, 65% 50 23 50 23 Meat and bone meal, 47% 50 23 50 23 Dicalciumphosphate 10 4 9 4 Ground limestone 16 7 14 6.3 DL-methionine 0.8 0.3 —— Stabilized yellow grease 101 45.7 110 49.4 Iodized salt 7 3 7 3Mineral and vitamin supplement Calcium pantothenate (mg) 5,000 5,000Manganese (g) 75 75 Organic arsenical supplement 0.1 0.1 Selenium (mg)90.8 90.8 Zinc (g) 30 30 Vitamin A (IU) 4,000,000 4,000,000 Vitamin D(IU) 1,000,000 1,000,000 Vitamin E (mg) 2,000 2,000 Vitamin K (mg) 2,0002,000 Choline (mg) 503,000 672,000 Niacin (mg) 20,000 20,000 Riboflavin(mg) 3,000 3,000 Vitamin B-12 (mg) 12 12 Total 2000.9 909.3 2000.1 909.5

F. Grower/Finisher Turkey Rations

TABLE 23 Grower Finisher (8-16 weeks) (16 weeks-market) Ingredients lbs.kgs. lbs. kgs. Ground yellow corn 1194 595 1490 677.2 Wheat middlings 5023 — — Alfalfa meal, 17% 25 11.3 25 11.3 Soybean meal, dehulled 570 259335 152.3 Meat and bone meal, 47% 50 23 50 23 Dicalcium phosphate 3214.5 23 10.5 Ground limestone 14 6 17 8 Stabilized yellow grease 45 20.745 20.7 CLA-ester 5 2.3 5 2.3 Iodized Salt 10 4.5 10 4.5 Mineral andvitamin supplements Calcium pantothenate (mg) 4,500 4,500 Manganese (g)30 30 Selenium (mg) 181.6 181.6 Zinc (g) 30 30 Vitamin (IU) 1,500,0007,500,000 Vitamin D (IU) 1,700,000 1,700,000 Vitamin E (IU) 10,00010,000 Biotin (mg) 100 100 Choline (mg) 388,000 417,000 Niacin (mg)46,000 48,000 Riboflavin (mg) 5,000 5,000 Vitamin B-12 6 6 Total 2000909.3 2000 909.3

G. Dry Dog Food Formula

TABLE 24 Ingredients Formula 1, % Formula 2, % Meat and bone meal, 50%CP 8.0 15.0 Fish meal, 60% CP, low fat 5.0 3.0 Soybean meal, 44% CP 12.0— Soybean meal, 50% CP — 19.0 Wheat germ meal, 25% CP 8.0 5.0 Skimmedmilk, dried 4.0 2.75 Cereal grains, mixed 51.23 — Corn, flaked — 23.25Wheat bran 4.0 — Wheat, flaked — 23.35 Animal fat 1.75 2.75 CLA-ester.25 .25 Steamed bone meal 2.0 — Brewers yeast 2.0 5.0 Fermentationsolubles, dehydrated 1.0 — Salt and trace minerals 0.5 0.5 Vitaminmixture 0.25 0.25 Ferric oxide 0.02 — Total 100.00 100.00

H. Semi-moist Dog Food Formulas

TABLE 25 Ingredients Formula 1, % Formula 2, % Soy flakes 30.9 33.5 Meatbyproducts, 70% moisture 32.0 — Meat and bone meal, dehydrated — 7.3Water — 25.6 Sugar 21.0 21.0 Calcium and phosphorous supplement 3.3 —Soybean hulls 3.1 3.1 Skimmed milk, dried 2.5 — Propylene glycol 2.1 2.1Sorbitol 2.0 2.0 Animal fat .75 3.95 CLA-ester .25 .25 Emulsifiers 0.9 —Potassium sorbate 0.35 0.35 Salt 0.6 0.6 Vitamins 0.25 0.25 Total100.000 100.000

EXAMPLE 11 Large-scale Preparation of CLA

This example illustrates a method of preparing free fatty acids of CLAon a pilot scale by the isomerization of safflower oil. 1000 kg of KOHwas dissolved in 2070 L of propylene glycol. The mixture was then heatedto 100° C. with stirring. Next, 2340 L of safflower oil was added andthe temperature was elevated to 150° C. for 3 hours. The mixture wasthen cooled and 1000 L of water and 1350 L of HCL was added. At thispoint, the solution separated into two layers, with the free fatty acidsas the top layer. The layers were separated and the bottom aqueous layerdiscarded. The top layer was washed with 1000 L of water containing 50kg of citric acid. The aqueous layer was discarded and the oil (CLA)containing layer was dried under vacuum.

EXAMPLE 12 Detection of Volatile Compounds

This example illustrates the detection of volatile organic compoundsusing head space capillary gas chromatography and mass spectroscopy.Three grams of CLA prepared by the method of Example 11 was purged withnitrogen at 100 ml/min at 70° C. in a sliff tube. Volatile compoundsreleased were absorbed on Tenex GR by the purge and trap technique. Theabsorbed compounds were then injected by a Perkin Elmer ATD injector onto a Hewlett Packard 5890/5970 GC/MSD gas chromatography system equippedwith a waxether column (J&W). Peaks were identified using Wileyreference search.

Table 27 shows the most predominant peaks and their relative abundance(area %). Eleven volatile compounds were identified in the sample,including pentane and hexane. Many of these compounds, including hexaneare undesirable in products for animal or human consumption. Theseresults demonstrate that CLA samples prepared by chemical conjugation ofoils contain undesirable volatile organic compounds.

EXAMPLE 13 Oxidation of CLA

This example demonstrates the oxidation of CLA over time. CLA wasprepared as described in Example 11. One sample was left in a test tubeat room temperature for 21 days. A second reference sample was stored at−30° C. The increase in pentane and hexane in both samples was measuredby the method described in Example 12. Results are shown in Table 28.The amount of both pentane and hexane present in the sample increased byapproximately two-fold after 21 days of storage at room temperature.This example demonstrated that CLA samples prepared by chemicalconjugation of oils oxidize over time to form undesirable volatileorganic compounds.

EXAMPLE 14 Production of Triacylglycerides

CLA was prepared according to the method of Example 11. The product wasthen distilled on a molecular distillation plant at 150° C. and apressure of 10⁻² mbar. Next, 1000 kg of the distilled product was mixedwith 97 kg of pure glycerol and 80 kg lipase. The reaction was allowedto proceed for 12 hours at 55° C. under vacuum and with stirring. Thetriacylglyceride product was distilled on a molecular distillationapparatus to remove unreacted fatty acids.

EXAMPLE 15 Oxidation of CLA Triacylglycerides

Aliquots of the product of Example 14 were placed in open dishes andstored under controlled conditions at 60° C. Antioxidants were added tosome of the samples in varying amounts. Antioxidants used were Controx(Grunau (Henkel), Illertissen, Del.), Herbalox (Kalsec, Kalamazoo,Mich.), Covi-OX (Grunau (Henkel), Illertissen, Del.), andalpha-tocopherol. Antioxidants were added at 0, 0.02, 0.05, and 0.10 %by weight.

The oxygen stability index (OSI) was measured using a method known inthe art (AOCS official method Cd 12b-92 using an OSI apparatus fromOmnion Instruments). Samples (5 g) were held in a thermostable bath anda stream of purified air was passed through the samples. The effluentair from the samples was bubbled through a vessel containing deionizedwater. The conductivity of the water is continually monitored over time.The OSI (the point of maximum change of the rate of oxidation) isdetermined mathematically. Results of the OSI measurements are shown inTable 29 and FIG. 2. The addition of alpha-tocopherol did notsignificantly increase the OSI value. Herbalox increased the value byapproximately 2-3 fold. Covi-OX and Controx increased the OSI values bya greater amount, approximately 4 and 6 fold, respectively. Thisexperiment demonstrated that the addition of certain antioxidantscontaining metal oxidant chelators can slow the oxidation of CLAcontaining compounds during storage.

EXAMPLE 16 Treatment with Absorbing Agents

A triacyglyceride of CLA was prepared as described in Example 14. Thesample was deodorized at 150° C. and 1 mm Hg for 3 hours. Next, 500 mlof the sample was treated with powdered silica. Silica was added to 2%and heated to 90-100° C. under vacuum for 30 minutes. The sample wasthen cooled and filtered.

TABLE 26 Retention Time Area % Volatile Compound 2.33 0.43 Pentane 2.520.64 Hexane 2.93 0.66 Heptane 5.65 0.75 2-Butenal 7.35 16.44 Ethanol8.81 5.85 3-Methyl Butanal 9.72 1.32 4-Methyl Pentanone 12.29 16.04Hexanal 15.55 8.26 Heptanal 16.63 1.29 2-Pentyl Furan 18.03 2.56 Octanal

TABLE 27 Volatile Compound Day zero (GC area × 1E6) Day 21 (GC area ×1E6) Pentane 52 105 Hexane 94 192

TABLE 28 OSI Values in the presence of antioxidants (standard deviation)alpha- Weight % Controx Herbalox Covi-OX tocopherol 0.00  24.27 (1.78)25.80 (4.88)  25.40 (5.69) 24.23 (3.32) 0.02  62.85 (1.24) 26.23 (1.37) 52.00 (2.73) 30.40 (2.02) 0.05 109.92 (2.38) 41.62 (1.71)  74.68 (8.00)38.97 (3.13) 0.10 161.50 (2.83) 66.95 (1.99) 111.38 (2.83) 40.47 (0.86)

EXAMPLE 17 Production of CLA with Alcoholate Catalysts

This example describes the production of CLA from safflower oil usingpotassium methylate as a catalyst. Distilled methyl ester of sunfloweroil (41.5 g) was placed in a reactor with 0.207 g methanol and 0.62 gpotassium methylate, and the reactor purged with nitrogen beforeclosing. The contents of the reactor were stirred while to 120° C. Thereaction was then allowed to proceed at 120° C. for 4 hours. the reactorwas then cooled to 80° C. and the contents were transferred to aseparating funnel and washed with hot distilled water and then with hotwater containing citric acid. The methylester was then dried undervacuum with moderate heat. The dried methyl ester was dissolved inisooctane and analyzed by GLC with a Perkin Elmer autosampler. Thecolumn was a highly polar fused silica type. the following program wasused:

Injection: Splitless at 250° C. Detection: Flame ionization detector at280° C. Carrier: Helium at psig. Oven program: 80° C.-130° C. (45°C./min.), then 1° C./min. to 220° C. and 220° C. throughout for 10 min.Column: WCOT FUSED SILICA 0.25 mm 100 m, CP-SIL 88 for FAME, df + 0.2.

The CLA obtained obtained consisted almost exclusively of the c9,t11 andt10,c12 isomers of CLA as shown in Table 30.

TABLE 29 CLA Produced by Isomerization with Potassium-Methylate FattyAcid Before Isomerization After Isomerization C 16:0 5.41 5.54 C 18:03.87 3.72 C 18:1 29.01 29.19 C 18:2, c9, c12 59.43 0.84 CLA, c9, c11 028.84 CLA, t10, c12 0 28.45 CLA, c9, c11 0 0.56 CLA, c10, c12 0 0.40CLA, t9, t11; t10, t12 0 0.27

EXAMPLE 18

Products Formed by Conjugation of Linolenic Acid

This example describes the products formed by conjugation of linolenicacid. Pure linolenic acid (Nu Check Prep.) was esterified and 1.08 gramsof the fatty acid methyl ester of linolenic was mixed with 43.0 mg ofpotassium methylate together with 10.8 mg methanol in atest tube. Thetest tube was purged with nitrogen and closed. A magnetic bar was usedfor stirring the mixture. The reaction was allowed to proceed at 120° C.for 3 hours. The sample was washed twice with water and once with citricacid and diluted into iso-octane for GLC analysis. The conditions werethe same as in example 17, except for the oven program and the column:

Oven program: 80° C.-160° C. (25° C./min.), then 5° C./min. to 220° C.and 220° C. throughout for 10 min. Column: WCOT FUSED SILICA 0.25 mm 100m, CP-SIL 5CB for FAME, df + 0.12.

The results (Table 31) demonstarte the existence of seven peaks of 1% ormore. Each of these peaks represents acids with 2 or 3 bonds in theconjugated position. It is not known if the fatty acids corresponding tothese peaks are found in nature and their possible effects are notknown.

TABLE 30 Relative Area Percentage of Unknown Isomers of ConjugatedLinolenic Acid Fatty Acid Before Conjugation After Conjugation C 18:399.53 2.43 unknown 0 55.82 unknown 0 0.98 unknown 0 2.03 unknown 0 3.79unknown 0 4.7 unknown 0 5.17 unknown 0 20.74

EXAMPLE 19 CLA Oxidation Products

This example describes the oxidation of CLA exposed to open air. A smallsample of methyl ester was prepared and stored in an open test tube for115 days at room temperature with free access to air. The CLA initiallypresent in the sample was completely broken down and transformed intofuran fatty acids and other unidentified derivatives. The relativecontent of the CLA sample before and after oxidation is shown in Table3. This chromatogram shows only relatively non-polar components. Achromatogram of the same sample was also run on a polar column. Theresults indicated the presence of a large number of short chain polarcomponents (i.e., breakdown products).

TABLE 31 CLA Composition Before and After Oxidation in Open Test Tubefor 115 days Fatty Acid Day 0 Day 115 C 16:0 1.36 1027 C18:1, t9 0 0.33C18:1, c9 4.14 3.19 C18:1, c11 0.13 0.10 Unknown 0 0.21 C18:2, c9, c120.06 0.03 CLA, c9, t11 7.68 0 CLA, t10, c12 7.63 0 CLA, c9, c11 0.11 0CLA, c10, c12 0.09 0 CLA, t9, t11; t10, t12 0.06 trace Unknown 0 0.26Unknown 0 0.26 Unknown 0.16 0.14

EXAMPLE 20 Energy Bar Containing CLA

This Example describes the formulation of a 48 gram nutritional barcontaining conjugated linoleic acid. The rice syrups and salt (binder)are slowly heated to 240° C. This mixture is removed from the heat andthe flavors are stirred in. This mixture is then poured over the rest ofthe ingredients (the dry mix), except the chocolate chips, and mixed ina mixing bowl. The chocolate chips are then added. Although anyconjugated linoleic acid supplement may be used (e.g., free fatty acids,alkyl esters, or triglycerides), it is preferred that the low volatilefatty acid triglycerides described in the previous examples are used inthis product. The recipe may be conveniently multiplied by 15.38 toprovide 15.38 servings in a single batch. The nutrients per serving forthis product are: calories —188.1; protein—11.68 g; carbohydrates—25.96g; dietary fiber—0.77 g; % calories from fat—22%; fat (total)—4.64 g;saturated fat—1.77 g; Viatamin A RE—0.51 RE; Vitamin C—13.69 mg; %calories from carbohydrates—54%. The components for a single serving arelisted in Table 32.

TABLE 32 Components of Nutritional Bar Component Amount (grams) Weight %Binder Brown rice syrup, 13.3328 27.78 BRSMC35CL84 Two fold vanillaextract 0.26015 0.54 flavor VDE Table salt 0.19511 0.41 Dry mix Suproplus nuggets 12.2193 25.46 Large crisp rice 2.60152 5.42 Rolled oats,dry 5.14766 10.72 Baker's Chocolate Chips, 2.60152 5.42 real semi-sweetLow volatile fatty acid CLA 0.97557 2.03 triglyceride Whey proteinconcentrate 1.30076 2.71 80, DMV Almond nuts toasted, whole 0.97557 2.03Soy grits, HS 2.92671 6.10 Vitamin mineral premix WE 0.58534 1.22 15625coating Westchester milk compound 4.87785 10.16

EXAMPLE 21 Energy Bar Containing CLA

This Example describes the formulation of a 48 gram nutritional barcontaining conjugated linoleic acid. This bar contains a convenient 3 gdaily dose of CLA. The rice syrups and salt (binder) are slowly heatedto 240° C. This mixture is removed from the heat and the flavors arestirred in. This mixture is then poured over the rest of the ingredients(the dry mix), except the chocolate chips, and mixed in a mixing bowl.If preferred, chocolate chips are then added. Although any conjugatedlinoleic acid supplement may be used (e.g., free fatty acids, alkylesters, or triglycerides), it is preferred that the low volatile fattyacid triglycerides described in the previous examples are used in thisproduct. The recipe may be conveniently multiplied by 13.1881 to provide13.1881 servings in a single batch. The nutrients per serving for thisproduct are: calories—185.7; protein—12.89 g; carbohydrates—24.64 g;dietary fiber—0.67 g; % calories from fat—20%; fat (total) —4.12 g;saturated fat—0.11 g; Viatamin A RE—0.60 RE; Vitamin C—15.93 mg; %calories from carbohydrates—52%. The components for a single serving arelisted in Table 32. The vitamins per serving are: Vitamin A—1068.21 IU;Vitamin A RE—0.60 RE; A—Carotenoid—0.60 RE; A—Retinol—0 RE;A—Beta-carotene—0 mcg; Thiamin B1—0.42 mg; Riboflavin B2—0.47 mg; NiacinB3—5.04 mg; Niacin equiv.—0.36 mg; Vitamin B6—0.56 mg; Vitamin B12—1.55mcg; Biotin—80.92 mcg; Vitamin C—15.93 mg; Vitamin E—Alpha Equiv. 0.04;Vitamin E IU—8.03; Vitamin E mg—0.04; folate—2.05 mcg; Vitamin K—3.78mcg.

TABLE 33 Components of Nutritional Bar Component Amount (grams) Weight %Binder Brown rice syrup, 15.5443 32.38 BRSMC35CL84 Two fold vanillaextract 0.3033 0.63 flavor VDE Table salt 0.22748 0.47 Dry mix Suproplus nuggets 14.2461 29.68 Large crisp rice 3.03303 6.32 Rolled oats,dry 6.00161 12.50 Low volatile fatty acid CLA 3.03303 6.32 triglycerideWhey protein concentrate 1.51652 3.16 80, DMV Soy grits, HS 3.41216 7.11Vitamin mineral premix WE 0.68243 1.42 15625 coating

What should be clear from above is that the present invention provides aconjugated linoleic acid composition of high purity that can be used inthe formulation of animal feeds and in food products suitable for humanconsumption.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmedicine, biochemistry, or related fields are intended to be within thescope of the following claims.

What is claimed is:
 1. A nutritional bar comprising a conjugatedlinoleic acid component, said conjugated linoleic acid componentcontaining less than 1% of the 8,10 and 11,13 isomers of conjugatedlinoleic acid.
 2. The nutritional bar of claim 1, wherein saidconjugated linoleic acid component contains less than 10 parts permillion total of volatile organic compounds.
 3. The nutritional bar ofclaim 1, wherein said conjugated linoleic acid component contains lessthan 5 parts per million total of volatile organic compounds.
 4. Thenutitional bar of claim 1, said nutritional bar further comprising avitamin supplement.
 5. The nutritional bar of claim 1, said nutritionalbar further comprising a carbohydrate component.
 6. The nutritional barof claim 1, said nutritional bar further comprising a protein component.7. The nutritional bar of claim 1, wherein said conjugated linoleic acidcomponent is provided as a triglyceride.
 8. A nutritional bar comprisinga conjugated linoleic acid component, said conjugated linoleic acidcomponent containing less than 100 parts per million total of volatileorganic compounds.
 9. The nutritional bar of claim 8, wherein saidconjugated linoleic acid component contains less than 10 parts permillion total of volatile organic compounds.
 10. The nutritional bar ofclaim 8, wherein said conjugated linoleic acid component contains lessthan 5 parts per million total of volatile organic compounds.
 11. Thenutritional bar of claim 8, wherien said conjugated linoelic acidcomponent contains less than less than 1% of the 8,10 and 11,13 isomersof conjugated linoleic acid.
 12. The nutitional bar of claim 8, saidnutritional bar further comprising a vitamin supplement.
 13. Thenutritional bar of claim 8, said nutritional bar further comprising acarbohydrate component.
 14. The nutritional bar of claim 8, saidnutritional bar further comprising a protein component.
 15. Thenutritional bar of claim 8, wherein said conjugated linoleic acidcomponent is provided as a triglyceride.
 16. A nutritional barcomprising a conjugated linoleic acid component, said conjugatedlinoleic acid component containing less than 100 parts per million totalof volatile organic compounds and less than 1% of the 8,10 and 11,13isomers of conjugated linoleic acid.
 17. The nutritional bar of claim16, wherein said conjugated linoleic acid component contains less than10 parts per million total of volatile organic compounds.
 18. Thenutritional bar of claim 16, wherein said conjugated linoleic acidcomponent contains less than 5 parts per million total of volatileorganic compounds.
 19. The nutitional bar of claim 16, said nutritionalbar further comprising a vitamin supplement.
 20. The nutritional bar ofclaim 16, said nutritional bar further comprising a carbohydratecomponent.
 21. The nutritional bar of claim 16, said nutritional barfurther comprising a protein component.
 22. The nutritional bar of claim16, wherein said conjugated linoleic acid component is provided as atriglyceride.